Microsphere-based composition for preventing and/or reversing new-onset autoimmune diabetes

ABSTRACT

AS-oligonucleotides are delivered in microsphere form in order to induce dendritic cell tolerance, particularly in the non-obese-diabetic (NOD) mouse model. The microspheres incorporate antisense (AS) oligonucleotides. A process includes using an antisense approach to reverse an autoimmune diabetes condition in NOD mice in vivo. The oligonucleotides are targeted to bind to primary transcripts CD40, CD80, CD86 and their combinations.

The present application claims the benefit of priority of U.S.Provisional Application Ser. No. 60/835,742, which was filed Aug. 4,2006 and U.S. Provisional Application Ser. No. 60/864,914, which wasfiled Nov. 8, 2006. The entire text of each of the aforementionedapplications is incorporated herein by reference.

BACKGROUND

The present disclosure generally relates to an antisense approach toprevent and/or reverse an autoimmune diabetes condition in NOD mice.This includes microsphere delivery of AS-oligonucleotides by injectionto achieve therapeutic effect that causes a negative modulatingactivity, particularly in the non-obese-diabetic (NOD) mouse model. Themicrospheres are fabricated using totally aqueous conditions, whichmicrospheres incorporate one or more antisense (AS) oligonucleotides.

Microparticles, microspheres, and microcapsules are solid or semi-solidparticles having a diameter of less than one millimeter, and may be lessthan 100 microns, which can be formed of a variety of materials,including synthetic polymers, proteins, and polysaccharides.Microspheres have been used in many different applications, primarilyseparations, diagnostics, and drug delivery.

A number of different techniques can be used to make these particlesfrom synthetic polymers, natural polymers, proteins and polysaccharides,including phase separation, solvent evaporation, emulsification, andspray drying. Generally the polymers form the supporting structure ofthese microspheres, and the drug of interest is incorporated into thepolymer structure. Exemplary polymers used for the formation ofmicrospheres include homopolymers and copolymers of lactic acid andglycolic acid (PLGA) as described in U.S. Pat. No. 5,213,812 to Ruiz,U.S. Pat. No. 5,417,986 to Reid et al., U.S. Pat. No. 4,530,840 to Ticeet al., U.S. Pat. No. 4,897,268 to Tice et al., U.S. Pat. No. 5,075,109to Tice et al., U.S. Pat. No. 5,102,872 to Singh et al., U.S. Pat. No.5,384,133 to Boyes et al., U.S. Pat. No. 5,360,610 to Tice et al., andEuropean Patent Application Publication Number 248,531 to SouthernResearch Institute; block copolymers such as such as Tetronic®908 andpoloxamer 407 as described in U.S. Pat. No. 4,904,479 to Illum; andpolyphosphazenes as described in U.S. Pat. No. 5,149,543 to Cohen et al.Microspheres produced using polymers such as these exhibit a poorloading efficiency and are often only able to incorporate a smallpercentage of the drug of interest into the polymer structure.Therefore, substantial quantities of these types of microspheres oftenmust be administered to achieve a therapeutic effect. In addition, thesepolymers typically are hydrophobic, negatively impacting the dissolutionof the drug of interest. Polymers typically used in this context includepolylactic glycolic acid (PLGA).

An objective for the medical community is the delivery of nucleic acidsto the cells in an animal for treatment of various diseases includingdiabetes. In many approaches, nucleic acids can be delivered to cells inculture (in vitro) relatively efficiently with the addition oftransfection agents. In addition, in vivo, the presence of endogenousnucleases results in a high rate of nucleic acid degradation whennucleic acid is delivered to animals.

In addition to protecting nucleic acid from nuclease digestion, anucleic acid delivery vehicle must exhibit low toxicity, must beefficiently taken up by cells and have a well-defined, readilymanufactured formulation. As shown in clinical trials, viral vectors fordelivery can result in a severely adverse, even fatal, immune responsein vivo. In addition, this method has the potential to have mutageniceffects in vivo. Delivery by complexing nucleic acids in lipid complexesof different formulations (such as liposomes or cationic lipidcomplexes) can have toxic effects. Complexes of nucleic acids withvarious polymers or with peptides have shown inconsistent results andthe toxicity of these formulations has not yet been resolved. Nucleicacids also have been encapsulated in polymer matrices for delivery, butin these cases the particles have a wide size range and theeffectiveness for therapeutic applications has not yet beendemonstrated. Such previous approaches can yield effects that are theopposite of a goal desired herein, including stimulation of the immunesystem. For example, when PLGA is incorporated into particles, theimmune system is stimulated by the presence of the PLGA.

Therefore, there is a need for addressing the issues in the delivery ofnucleic acids, and there is an ongoing need for development ofmicrospheres and new methods for making microspheres. Details regardingmicrospheres, especially details regarding their preparation andproperties, are found in U.S. Pat. No. 6,458,387 to Scott et al., U.S.Pat. Nos. 6,268,053, 6,090,925, 5,981,719 and 5,599,719 to Woiszwillo etal., and U.S. Pat. No. 5,578,709 to Woiszwillo and US Patent ApplicationPublication No. 2006/0024240 to Brown et al. These and all referencesidentified herein are incorporated by reference hereinto.

SUMMARY

In accordance with the present disclosure, oligonucleotides aredelivered as microspheres. It is believed that such a delivery approachprevents access of the nucleases to the nucleic acids within themicrosphere. Microsphere delivery of antisense (AS) oligonucleotides iscarried out in order to induce dendritic cell tolerance, particularly inthe NOD mouse model. The microspheres are fabricated using aqueousconditions such that antisense (AS) oligonucleotides are incorporated.These microspheres are used to inhibit gene expression and to preventand/or reverse an autoimmune diabetes condition in NOD mice in vivo.

In a one aspect of the disclosure, three AS-oligonucleotides targeted tothe CD40, CD80 and CD86 transcripts are synthesized, and an aqueoussolution of the oligonucleotide mixture is prepared and combined with anaqueous polymer solution. Microspheres containing the oligonucleotidesare formed, and these are delivered to the NOD mice by injection.

In one aspect of the disclosure, there is provided a method forreversing type 1 diabetes in a mammal comprising administering amicrosphere composition wherein microspheres in the composition compriseoligonucleotides that are antisense to and targeted to bind to primarytranscripts selected from the group consisting of CD40, CD80 and CD86primary transcripts and combinations thereof. The oligonucleotides canbe selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 or SEQID NO:3 and combinations thereof, or indeed any other oligonucleotidesthat target CD40, CD80 and CD86.

Another aspect of the disclosure is directed to a method of protectingbeta cells of the pancreas of a mammal from autoimmune destruction,comprising injecting into the mammal a microsphere composition, whereinthe microspheres in the composition comprise oligonucleotides that areantisense to and targeted to bind to primary transcripts selected fromthe group consisting of CD40, CD80 and CD86 primary transcripts andcombinations thereof.

Another aspect is a method of decreasing T-cell-mediated inflammation ofthe pancreas and/or pancreatic beta cell death in a mammal comprisingadministering to the mammal a microsphere composition, wherein themicrospheres in the composition comprise oligonucleotides that areantisense to and targeted to bind to primary transcripts selected fromthe group consisting of CD40, CD80 and CD86 primary transcripts, andcombinations thereof, wherein the composition is administered in anamount effective to ameliorate the symptoms of Type 1 diabetes in themammal. In more defined aspects, the composition is administered afterclinical onset of Type 1 diabetes. In alternative aspects, thecomposition is administered prior to clinical onset of Type 1 diabetes.In these therapeutic aspects, the administration of the compositionnormalizes blood glucose levels in the mammal as compared to the bloodglucose levels of the mammal prior to administration.

The administration of the composition may regenerate the beta cellpopulation of the mammal or halt the further deterioration of the betacell population or both.

The composition may be administered in any form and in certain exemplaryaspects is administered as an injectable form. In specific aspects, thecomposition is administered in combination with insulin. Where acombination therapy is used, the insulin may be administered prior to,concurrently with, or after administration of the microspherecomposition.

Additional aspects are directed to methods of preserving residual betacell mass in a subject with new-onset or preclinical autoimmune diabetescomprising administering to the subject a composition containingmicrospheres comprising oligonucleotides that are antisense to andtargeted to bind to CD40, CD80 and CD86 primary transcripts, whereinadministration of the composition maintains the beta cell mass of themammal to at least about 15% of the mass present prior to diabetesonset. The subject may be a human subject. The subject may be a humanchild. The treatment method may involve repeated administration of thecomposition and the repeated administration increases the beta cell massof the mammal.

In particular defined methods, 30% and as much as 70% w/w of themicrospheres is oligonucleotide. Such compositions typically maycomprise a ratio in the microsphere composition of antisenseCD40:antisense CD80: antisense CD86 of 1:1:1.

These and other aspects, objects, features and advantages of the presentdisclosure, including the various combinations, will be apparent fromand clearly understood through a consideration of the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

In the course of this description, reference will be made to theattached drawings, wherein:

FIGS. 1 a and 1 b are scanning electron micrographs of microspheres ofAS-oligonucleotides and poly-L-lysine polycation.

FIGS. 2 a and 2 b are graphs showing the properties of a microspherepreparation according to the disclosure. FIG. 2 a is graph showing thesize distribution of a preparation of microspheres. FIG. 2 b shows agraph of the surface charge of a preparation of microspheres.

FIG. 3 is a RP-HPLC chromatogram of the oligonucleotides afterdeformulation of microspheres.

FIG. 4 is a plot showing the prevention of diabetes in NOD mice treatedmultiple times with antisense oligonucleotide microspheres (AS-MSP) ofthe disclosure compared to animals treated with scrambledoligonucleotides microspheres or with the PBS vehicle alone.

FIG. 5 is a plot showing the prevention of diabetes in NOD mice treatedonce with AS-MSP of the disclosure compared to animals treated withscrambled oligonucleotide microspheres or with the PBS vehicle alone.

FIGS. 6 a-6 d are light micrographs of pancreatic tissue sections fromcontrol NOD mice stained with haemotoxylin and eosin (FIG. 6 and c; H+E)or for insulin (FIGS. 6 b and 6 d).

FIGS. 7 a-7 d are light micrographs of pancreatic tissue sections fromAS-MSP treated NOD mice stained with haemotoxylin and eosin (FIG. 7 aand c; H+E) or for insulin (FIGS. 7 b and 7 d).

FIG. 8 shows a FACS analysis of T cells obtained from mice treated withthe AS-MSP of the disclosure or from control animals.

FIG. 9 shows plots of relative fluorescent intensity (RFI) demonstratingthe proliferation of T cells from animals treated with AS-MSP andcultured with splenocytes according to the disclosure.

FIG. 10 shows plots of RFI demonstrating the proliferation of T-cellsfrom AS-MSP treated, diabetes-free NOD mice in the presence of syngeneicirradiated splenocytes and ovalbumin in vitro.

FIG. 11 shows plots of RFI demonstrating the suppressed proliferation ofT-cells from AS-MSP-treated, diabetes-free NOD mice in the presence ofsyngeneic islet lysate in vitro.

FIG. 12 is a plot of blood glucose levels from new-onset diabetic micetreated with either microspheres containing antisense or scrambledoligonucleotides.

FIG. 13A shows a timeline for the experiments with mice having new-onsetdiabetes, and FIGS. 13B and 13C are plots of mean blood glucose levelsfrom new-onset diabetic mice treated with either AS-MSP or controls.

FIG. 14A-C shows reversal of the type-1 diabetes phenotype in NOD mice.These figures show that upon administration of AS-MSP the blood glucoselevels of the mammals return to normal within 15 days (normal levels areshown by the dashed line at approx 200 mg/dL) and remain at normal evenafter AS-MSP administration is stopped (day 30).

FIG. 15 Model depicting therapeutic reversal of autoimmune diabetes.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

As required, detailed embodiments of the present disclosure aredisclosed herein; however, it is to be understood that the disclosedembodiments are merely exemplary of the disclosure, which may beembodied in various forms. Therefore, specific details disclosed hereinare not to be interpreted as limiting, but merely as a basis for theclaims and as a representative basis for teaching one skilled in the artto variously employ the present disclosure in virtually any appropriatemanner.

Type I diabetes is an autoimmune disorder where there is a progressiveinflammation of the pancreas, and specifically, the endocrineinsulin-producing beta cells. Before onset, the inflammation firstrenders the endocrine beta cells dysfunctional. A single injection of amicrosphere formulation considerably delays disease onset in thenon-obese diabetic (NOD) mouse model of human autoimmune (type 1)diabetes. Although not wishing to be bound by any particular theory, itis believed the microspheres are taken up by resident and migratingdendritic cells at the site of injection and then move into the proximallymph nodes before onset of the disease. It is also believed that adecreased proliferation of T-cells targeted to putative beta cellantigens in vitro occurs in treated recipients. An increase may occur inthe prevalence of CD4+CD25+ putative T regulatory cells inimmunodeficient NOD-scid mice reconstituted with syngeneic T-cells anddendritic cells and then administered the microspheres. Thus, amicrosphere-based therapeutic composition can modulate dendritic cellactivity and mobilize regulatory networks for prophylaxis.

It would be desirable to have a treatment that would prevent the onsetof diabetes. It would also be desirable to have a therapeuticcomposition that would arrest or reverse the disease after clinicalonset when a substantial number of beta cells have been destroyed.Repeated administration into new-onset diabetic mice normalizeshyperglycemia and reverses the disease. Reversal typically indicateshaving the individual, such as a human or other mammal, exhibit nearnormalization of blood glucose levels. Without being bound by anyparticular theory, it is believed that during “reversal”,disease-induced T-cell inflammation and cell death are resisted.

One embodiment reverses autoimmune insulin-dependent diabetes byformulating and injecting antisense (AS)-oligonucleotide microspheresdescribed herein, targeting the transcripts of CD40, CD80 and CD86.Specific examples of antisense oligonucleotides directed against thetranscripts are disclosed in the Examples hereof. It will be understoodthat other antisense oligonucleotides may be designed to be effective inbinding the CD40, CD80 and CD86 transcripts to achieve the effectsdescribed herein. It will also be understood that such oligonucleotidesmay incorporate modifications known in the art including, but notlimited to, thioation, methylation and methoxyethylation and that thelocation and number of such modifications may be varied to achieve anoptimal effect. These oligonucleotides are designed to induce immunetolerance that results in the reversal of the destruction of the insulinproducing beta cells in the NOD mouse model.

Type 1 diabetes is manifested by the autoimmune destruction of thepancreatic insulin-producing beta cells in the NOD mouse, as well as inhumans. At the time of clinical onset, humans typically have 10-20% orless of residual beta cell mass. Sparing of any of this residual masscan result in remaining insulin levels which are adequate to regulateglucose levels. In addition, reversing the destruction of beta cells mayresult in the partial regeneration of the beta cell population. Theoligonucleotide-containing microparticles of the present disclosure areprovided to interfere with the autoimmune destruction of the beta cells.

It will be appreciated that dendritic cells (DC) can be activated to bepotent antigen-presenting cells found in all tissues and which arepresent under the skin. These antigen-presenting dendritic cellsfunction as triggers of the immune response, including autoimmuneresponses, through the activation of T-cells, particularly in lymphnodes. Although not wishing to be bound by theory, CD40, CD80 and CD86are believed to be important for the autoimmune response, and thedownregulation of these molecules is thought to promote autoimmunehyporesponsiveness. In addition, certain cytokines, such as interferonsand interleukins, are reduced as a result of the hyporesponsiveness.

In making the microspheres that are used for treatment of autoimmunediabetes in mice, one, two or three AS-oligonucleotides may be dissolvedin aqueous solution and combined with water soluble polymer(s) and apolycation. The solution typically is incubated at about 60-70° C.,cooled to about 23° C., and the excess polymer is removed.

The nucleic acids typically comprise between about 30 and about 100weight percent of the microspheres and have an average particle size ofnot greater than about 50 microns, typically not greater than about 20microns, and can be not more than about 10 microns. Typically, they areprepared as follows. An aqueous solution of the oligonucleotide oroligonucleotides is prepared. When microspheres containing threeoligonucleotides are to be prepared, aliquots from three oligonucleotidesolutions are combined. Each solution contains one of these threeoligonucleotide types. The final solution containing oligonucleotidestypically contains about 10 mg/ml of oligonucleotide.

In specific examples, the microsphere formulation contains 65%, 70%,75%, 80%, 85%, 90% w/w or greater load of oligonucleotides. In suchembodiments, the compositions have a poly-L-lysine content of 6-10% w/w.in addition the moisture content of the microspheres varies and can beapproximately 4%. The oligonucleotides are present in a ratio of 1:1:1of antisense CD40:antisense CD80:antisense CD86.

These are combined with aliquots of a 10 mg/ml stock solution ofpolycation. Examples of polycations are poly-lysine and poly-ornithine.Others include polyethyleneimine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, vinylamine, and derivatives ofpositively-charged polysaccharides, such as positively charged chitosan,and combinations thereof. The polycation solution can be at volumetricratios of polycation:oligonucleotide of from about 1:1 to about 4:1.Commonly used polycations include poly-L-lysine•HBr (up to 70,000Daltons available from Bachem) and poly-L-omithine•HBr (e.g. 11,900Daltons available from Sigma).

Polymer solutions also are prepared. These can function asphase-separation enhancing agents. Examples of suitable polymers includelinear or branched polymers, copolymers and block copolymers. Thesepolymers can be water soluble, semi-water soluble, water-miscible, orsoluble in a water-miscible solvent. Examples of polymers includepharmaceutically acceptable additives such as polyethylene glycol (PEG)of various molecular weights, such as PEG 200, PEG 300, PEG 3350, PEG8000, PEG 10000, PEG 20000, etc. and poloxamers of various molecularweights such as poloxamer 188 and Pluronic F127 or Pluronic F68. Acommonly used polymer is polyvinylpyrrolidone (PVP). Another polymer ishydroxyethylstarch. Other amphiphilic polymers can also be used alone orin combinations. The phase-separation enhancing agent can also be anon-polymer such as a mixture of propylene glycol and ethanol.

In a typical embodiment, a polymer solution of polyvinyl pyrrolidoneand/or of polyethylene glycol may be prepared and combined with theother solutions. Heating, cooling, centrifuging and washing multipletimes provide an aqueous suspension which typically is frozen andlyophilized to form a dry powder of microspheres comprisingoligonucleotide and polycation.

The microspheres are suitable for in vivo delivery by an injectableroute, including intravenous, intramuscular, subcutaneous,intraperitoneal, intrathecal, epidural, intra-arterial, intra-articularand the like. Other delivery routes that can be practiced include suchas topical, oral, rectal, nasal, pulmonary, vaginal, buccal, sublingual,transdermal, transmucosal, otic or intraocular.

Without being bound by any particular theory, it is believed thatmicrospheres containing the antisense oligonucleotides exemplifiedherein down-regulate cell surface molecules CD40, CD80 and CD86. Themicrospheres are injected and dendritic cells are believed to activelyuptake the oligonucleotide microspheres. These oligonucleotides suppressthe expression of cell surface cell molecules CD40, CD80 and CD86 indendritic cells. The administration of these antisense oligonucleotidemicrospheres after development in the NOD mouse effectively reversesdiabetes.

The following Examples illustrate certain features and advantages of thedisclosure in order to further illustrate the disclosure. The Examplesare not to be considered limiting or otherwise restrictive of thedisclosure.

EXAMPLE 1

Three AS-oligonucleotides targeted to the CD40, CD80 and CD86 primarytranscripts were synthesized. The AS-oligonucleotide sequences used inthis Example are, with asterisks indicating sites of thioation in thebackbone:

Seq ID 1: CD 40-AS: 5′C*AC* AG*C C*GA* GG*C* AA*A GA*C* AC*C A*T*G C*AG*GG*C* A-3′ Seq ID 2: CD80-AS: 5′-G*GG* AA*A G*CC* AG*G A*AT* CT*A G*AG*CC*A A*TG G*A-3′ Seq ID 3: CD86-AS: 5′-T*GG* GT*G C*TT* CC*G T*AA* GT*TC*TG* GA*A C*AC* G*T*C_3′

An aqueous solution of the oligonucleotide mixture was prepared bycombining aliquots of three oligonucleotide solutions, each of whichcontained one type of oligonucleotide, to form a 10 mg/ml solution ofthe three types of oligonucleotides. A 10 mg/ml solution ofpoly-L-lysine•HBr in deionized water (poly-L-lysine•HBr up to 70,000Daltons, by Bachem, King of Prussia, Pa.) was prepared. Thepoly-L-lysine•HBr was added to the oligonucleotides solution at avolumetric ratio of 1:1. The mixture was vortexed gently. A 25% polymersolution containing 12.5% PVP (polyvinyl pyrrolidone, 40,000 Daltons,Spectrum Chemicals, Gardena, Calif.) and 12.5% PEG (polyethylene glycol,3,350 Daltons, Spectrum Chemicals, Gardena, Calif.) in 1M Sodium Acetate(Spectrum, Gardena, Calif.) at pH5.5 was added in a 2:1 volumetric ratioas follows: 0.75 ml of AS-oligonucleotides, 0.75 ml ofpoly-L-lysine•HBr, 3.0 ml of PEG/PVP, and a total volume of 4.50 ml.

The batch was incubated for 30 minutes at 70° C. and then cooled to 23°C. Upon cooling, the solution became turbid and microspheres wereformed. The suspension was then centrifuged, and the excess PEG/PVP wasremoved. The resulting pellet was washed by resuspending the pellet indeionized water, followed by centrifugation and removal of thesupernatant. The washing process was repeated three times. The aqueoussuspension was frozen and lyophilized to form a dry powder ofmicrospheres comprising oligonucleotide and poly-L-lysine.

FIGS. 1 a and b present representative scanning electron micrographs(SEM) of 1:1 poly-L-lysine: oligonucleotide ratio microspheres at twodifferent magnifications. Microspheres, 0.5-4 μm in size, with anaverage particle size of approximately 2.5 μm were fabricated. FIG. 2 ashows the size distribution of one preparation of microspheres madeaccording to the disclosure as revealed by laser light scattering. FIG.2 b shows the determination of the surface charge of a microspherepreparation (Zeta potential) by light scattering. FIG. 3 shows a reversephase (RP) HPLC method used to quantitate the loading and assess theintegrity of the antisense oligonucleotide components of themicrospheres after deformulation. Microspheres were formulated usingCD86, CD40, CD80 oligonucleotides and poly-L-lysine (PLL; MW 30-70 kD).The microspheres were then deformulated using competitive displacementof the DNA oligonucleotides from the PLL by poly-L-aspartic acid (PAA).PAA was selected as a polyamino acid reagent that does not absorb at 260nm and does not interfere with quantification of oligonucleotides at 260nm. In RP-HPLC profiles such as FIG. 3, the area under each peak isproportional to amount of each oligonucleotide loaded into themicrosphere. As shown in FIG. 3, the peak heights indicate approximatelyequal loading of each oligonucleotide into microspheres. The loading ofoligonucleotides into microspheres was calculated to be from about 65%to about 80% by weight. FIG. 3 also shows that the integrity of theoligonucleotides was not affected by the microsphere formulationprocess, as indicated by the narrow distribution of the peaks afterdeformulation.

EXAMPLE 2

In this Example, the results of tests that cover prevention aspects ofthe disclosure are shown. As shown in FIG. 4, a single AS-MSPadministration into NOD mice at 5-8 weeks of age delays diabetes onset.Two groups of NOD female mice (5-8 weeks old) were given a singlesubcutaneous injection of antisense-oligonucleotides formulated intomicrospheres of the disclosure (AS-MSP). The formulation was injected ininjected in an amount considered to contain 50 μg of a 1:1:1 mixture ofeach antisense oligonucleotide (anti-CD40, anti-CD80 and anti-CD86).Other groups of mice were injected with scrambled sequence microspheres(SCR-MSP) or PBS vehicle (control NOD). Blood glucose was measuredweekly via tail vein puncture. Diabetes was confirmed after twoconsecutive readings of >280-300 mg/dL. FIG. 4 shows the cumulativesurvival of two independently-treated cohorts.

FIG. 5 shows that multiple AS-MSP administration into NOD mice at 5-8weeks of age prevents diabetes onset. NOD female mice (5-8 weeks old)were given eight consecutive single subcutaneous injections (onceweekly) of antisense oligonucleotide formulated into microspheresaccording to the disclosure. Injections (50 μg of a 1:1:1 mixture ofeach antisense oligonucleotides or scrambled oligonucleotides) weregiven once weekly for eight weeks and stopped at week 13. Other groupsof mice were injected with scrambled sequence microspheres (SCR-MSP) orPBS vehicle (control NOD). FIG. 5 shows the cumulative survival oftreated animals.

FIGS. 6 a and 6 b show sections of pancreatic tissue from mice thatreceived no treatment and thus progress spontaneously to autoimmunity(diabetic NOD mice) stained with haemotoxylin and eosin (H+E; FIG. 6 a)or stained for insulin (FIG. 6 b). FIG. 6 c and 6 d show sections ofpancreatic tissue from mice treated with SCR-MSP formulations(injections started in parallel with the groups treated with specificAS-MSP). These sections were also stained with haemotoxylin and eosin(H+E; FIG. 6 c) or stained for insulin (FIG. 6 d). The SCR-MSP mice alldeveloped diabetes.

FIGS. 7 a and 7 b shows sections of pancreatic tissue from mice treatedwhen less than 8 weeks of age (prevention model) and treated with theantisense microspheres of the disclosure stained with haemotoxylin andeosin (H+E; FIG. 7 a) or stained for insulin (FIG. 7 b).

As shown in FIG. 8, T-cells from AS-MSP treated, NOD mice exhibitincreased prevalence of Foxp3+ CD25+ putative T_(reg) cells. FIG. 8Ashows the gating used for FACS analysis. FIG. 8B shows percentages ofFoxp3+ CD25+ T-cells that were enriched from the spleen and FIG. 8C thepercentages from the pooled lymph nodes for ASMSP-treated diabetes-freemice selected at random from the ASMSP diabetes-free cohort or from orfrom animals treated with scrambled sequence microspheres (SCR-MSP) ortreated with PBS vehicle.

FIG. 9 shows that T-cells from ASMSP-treated diabetes-free NOD miceproliferate when co-cultured with allogeneic splenocytes. T-cells fromdiabetes-free NOD mice treated with ASMSP were obtained over enrichmentcolumns and co-cultured with γ-irradiated splenocytes from Balb/c,C57BL6 or syngeneic diabetes-free NOD mice (10 weeks of age).Proliferation was measured four days later using the Cyquant reagent.Spl refers to allogeneic irradiated splenocytes.

As shown in FIG. 10, T-cells from ASMSP-treated, diabetes-free NOD miceproliferate in the presence of syngeneic irradiated splenocytes andovalbumin in vitro. T-cells were enriched from the spleen or the pooledlymph nodes of ASMSP-treated diabetes-free mice selected at random fromthe ASMSP diabetes-free cohort.

FIG. 11 shows that T-cells from ASMSP-treated, diabetes-free NOD miceexhibit suppressed proliferation in the presence of syngeneic isletlysate in vitro. T-cells were enriched from the spleen or the pooledlymph nodes of ASMSP-treated diabetes-free mice selected at random fromthe ASMSP diabetes-free cohort as described in FIG. 4. Irradiated NODsplenocytes (from diabetes-free 10 week-old NOD mice) were used asantigen-presenting cells and parallel cultures were pulsed with NIT-1lysate (1 μg/well)(or PBS vehicle).

A major concern for eventual translation of diabetes-suppressivetherapies into human trials is the antigen specificity (and thereforethe cell specificity) of the treatment approach and whether thetreatment confers global and non-specific suppression. To address theseissues, randomly-selected diabetes-free mice were euthanised from thecohorts shown in FIG. 4 to ascertain the proliferation of splenic andlymph node T-cells to alloantigen, nominal antigen (in the form ofintact ovalbumin) and to syngeneic beta cell-derived antigen in the formof cell lysate from the NOD derived insulinoma cell line NIT-1. Whileinsulin and glutamic acid decarboxylase (GAD) are viable candidateautoantigens with mechanistic and teleologic involvement, the nature ofthe initiating autoantigen remains unclear. Nevertheless, it isreasonable to consider that it should be beta-cell resident. Therefore,the NIT-1 cell line which derives from an NOD insulinoma was used as asource of beta cell antigen in cocultures of T-cells from diabetes-freeNOD mice treated with the AS-MSP to determine the possibility ofantigen-specific hyporesponsiveness. From these studies, it was seenthat T-cell proliferation to nominal and alloantigen is maintainedwhereas there is T-cell hypoproliferation in cocultures with NIT-1 celllysate.

Furthermore, ascertaining the cytokine profile in the co-culturesupernatants, we observed a significant decrease in TNFα production byT-cells from AS-MSP-treated, diabetes-free NOD mice even in the presenceof NIT-1 lysate. Although IFNγ production was slightly decreased in theco-cultures of T-cells from the AS-MSP-treated mice, it was notstatistically-distinguishable from the co-cultures with T-cells fromPBS-treated mice in the presence of NIT-1 lysate. The assay, finally,could not detect the presence of IL-4, IL-10 or TGFfβ in thesupernatants.

EXAMPLE 3

The ability of antisense oligonucleotide microspheres to reverse thesymptoms of diabetes in early onset NOD mice was also tested. A timelinefor these experiments is shown in FIG. 13A. NOD mice that had earlyonset were selected by testing blood glucose levels and identifyinganimals that had a blood glucose level greater than 400 mg/dL. Theselected animals were given insulin pellets to normalize blood glucoselevels to below 300 mg/dL. The insulin was withdrawn and a series ofparenteral injections of microspheres was started. Six animals wereinjected twice weekly with microspheres containing the CD40, CD80 andCD86 antisense oligonucleotides. A further ten animals were injectedwith microspheres containing a mixture of oligonucleotides withscrambled sequences that are not directed against CD40, CD80 and/orCD86. Each injection for both groups of animals contained 50 μg ofoligonucleotides in microspheres in 100 microliters of injectionsolution. Two of the animals in the scrambled group were euthanizedbefore the end of the experiment due to poor physical condition. Afterthe commencement of the injection protocol, blood glucose levels weresampled twice weekly. The animals were non-fasting during theexperiment. The results are plotted in FIG. 12, wherein the indicator(1) signifies insulin pellet installation and indicator (2) signifiesinsulin pellet removal and initiation of MSP injections twice weekly. Itis noted that the maximum blood glucose value reported in FIG. 12 is 700mg/dL, which corresponds to the maximum reading of the meter used, itbeing understood that a 700 mg/dL data point indicates a blood glucosereading of 700 mg/dL or higher, All animals in the group that receivedthe microspheres containing the mixture of CD40, CD80, CD86 antisenseoligonucleotides (ASMSP1 through ASMSP6) showed significantly lowerglucose levels than the animals that received the microspheres withscrambled oligonucleotides (SCRMSP1 through SCRMSP10). Furthermore, fourof six animals in this ASMSP group showed a blood glucose level below400 mg/dL, typically considered to be a threshold indicator of diabetesonset.

In FIG. 13A, the timeline for the experiments is shown. The meannon-fasting blood glucose (FIG. 13B) and the mean fasting blood glucoselevels for each group are plotted (FIG. 13C) (+/− SEM). In some mice,ASMSP administration was withdrawn as shown in FIG. 13A. As shown inFIG. 13B and 13C, multiple rounds of AS-MSP administration intonew-onset diabetic NOD female mice improves blood glucose levels andresult in stable fasting euglycemia even after AS-MSP withdrawalrelative to untreated animals (control), animals treated with PBS oranimals treated with scrambled oligonucleotides (SCR-MSP) microspheres.

FIGS. 7 c and 7 d show sections of pancreatic tissue from NOD mice thatwere treated with antisense formulations after onset of diabetes andshowed reversal of the disease. The sections are stained withhaemotoxylin and eosin (H+E; FIG. 7 c) or stained for insulin (FIG. 7d).

3 different AS-oligonucleotides can be incorporated into PROMAXXmicrospheres and such microspheres can be used as a composition toprevent and/or reverse new onset autoimmune diabetes viaimmunoregulatory dendritic cell induction. Indeed, a single injection ofthe composition delayed disease onset and repeated administration intonew-onset diabetic mice normalized hyperglycemia, suggesting reversal ofdisease. In these studies, insulin was administered daily until bloodglucose fell below 300 mg/dL. Insulin then was stopped whereupon AS-MSPwere administered subcutaneously. In an exemplary dosing regiment, theanimals were administered 2 mg AS-MP per kg body weight two times a weekfor 3-4 weeks. The diabetes-free NOD mice were monitored.

In FIG. 14A-C it is demonstrated that administration of AS-MSP to NODmice returns the blood glucose levels of said mice to normal levels andthe normalization of said blood glucose level is maintained for anextended period of time. As shown in FIG. 14B and 14C, AS-MSP wasadministered between days 0-30 after insulin administration was stopped.The blood glucose level returned to normal by day 15 post insulin stopand remained at a normal level until the end of the monitoring period(day 55).

A diagram showing the impact of therapeutic reversal of autoimmunediabetes is show in FIG. 15. If PROMAXX treatment were administered atthe new onset “honeymoon” shown in FIG. 15, it is predicted that therewould be a preservation of the 10-20% beta cells that remain functional,thereby leading to a control of the diabetes and reducing the dependenceof the patient on insulin.

It will be understood that the embodiments of the present disclosurewhich have been described are illustrative of some of the applicationsof the principles of the present disclosure. Numerous modifications maybe made by those skilled in the art without departing from the truespirit and scope of the disclosure. Various features which are describedherein can be used in any combination and are not limited to precisecombinations which are specifically outlined herein.

1. A method for reversing Type 1 diabetes in a mammal comprisingadministering a microsphere composition directly to the mammal, whereinmicrospheres in said composition comprise oligonucleotides that areantisense to and targeted to bind to primary transcripts selected fromthe group consisting of CD40, CD80 and CD86 primary transcripts, andcombinations thereof, and wherein administration of the microspherecomposition promotes autoimmune hyporesponsiveness in the mammal.
 2. Themethod of claim 1, wherein said oligonucleotides are selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 andcombinations thereof.
 3. The method of claim 1, wherein said compositionis administered after clinical onset of Type 1 diabetes.
 4. The methodof claim 1, wherein said composition is administered prior to clinicalonset of Type 1 diabetes.
 5. The method of claim 1, whereinadministration of said composition normalizes blood glucose levels insaid mammal as compared to the blood glucose levels of said mammal priorto administration.
 6. The method of claim 1, wherein said methodcomprises administration of a composition containing microspheres thatcomprising oligonucleotides that are antisense to and targeted to hindto CD40, CD80 and CD86 primary transcripts.
 7. The method of claim 1,wherein said composition is administered as an injectable form.
 8. Themethod of claim 1, wherein said composition is administered incombination with insulin.
 9. The method of claim 8, wherein said insulinis administered prior to, concurrently with, or after administration ofsaid microsphere composition.
 10. The method of claim 1, wherein 70% w/wof said microspheres is oligonucleotide.
 11. The method of claim 10,wherein the ratio in said microsphere composition of antisense CD40:antisense CD80: antisense CD86 is 1:1:1.